Western Blot Analysis of Parasite Proteins

Protein samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Protran) in a tankblot device (Bio-Rad) using transfer buffer (0.192 M Glycine, 0.1% SDS, 25 mM Tris) with 20% methanol. Blocking of membranes and dilutions of antibodies were done in PBS containing 5% skim milk. Washing steps were done with PBS. Primary antibodies were applied in the following dilutions: mouse anti-GFP (Roche) 1:1000; rabbit anti-GFP (Thermo) 1:2000; rat anti-mCherry, 1:1000 (Chromotek); rabbit anti-SERA5, 1:2000 (newly raised); rabbit anti-REX3, 1:2000 (newly raised); mouse anti-SBP1N, 1:2500 (newly raised); rabbit anti-aldolase, 1:4000 (newly raised); mouse anti-HSP101, 1:1000 [7 (link)]; rabbit anti-DHFR (Abcam), 1:1000; rat anti-HA (Roche) 1:4000. Horseradish peroxidase-conjugated secondary antibodies used were goat anti-rat (Dianova) and goat anti-mouse (Dianova) and diluted 1:3000 as well as donkey anti-rabbit (Dianova) 1:2500 and applied after three washes. Immunoreactions were detected by enhanced chemiluminiscence (Bio Rad/ Thermo) and detected on CEA RP NEW x-ray films (Agfa). For quantification of Western blot signals, band intensities were measured with a Chemi Doc XRS imaging system (Bio-Rad) and densitometry analyses were done with Image Lab Software 5.2 (Bio-Rad). Data are representative of three independent experiments.